respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]

Contents lists available at ScienceDirect

Respiratory Investigation
journal homepage: www.elsevier.com/locate/resinv

Original article

Pathogen proles and molecular epidemiology

of respiratory viruses in Japanese inpatients
with community-acquired pneumonia
Daisuke Kuraia,1, Yoshiko Sasakib,1, Takeshi Sarayaa, Haruyuki Ishiia,
Hiroyuki Tsukagoshib, Kunihisa Kozawac, Akihide Ryoc, Taisei Ishiokad,
Makoto Kurodae, Kazunori Oishif, Hajime Takizawaa,n, Hirokazu Kimurac,f,nn
a

Department of Respiratory Medicine, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi,
Tokyo 181-8611, Japan
b
Gunma Prefectural Institute of Public Health and Environmental Science, 378 Kamioki-machi, Maebashi-shi,
Gunma 371-0052, Japan
c
Department of Microbiology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku,
Yokohama-shi, Kanagawa 236-0004, Japan
d
Takasaki City Health Center, 5-28 Takamatsu-cho, Takasaki-shi, Gunma 370-0829, Japan
e
Pathogen Genomics Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
f
Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi,
Tokyo 208-0011, Japan

art i cle i nfo

ab st rac t

Article history:

Background: The etiological prole of viruses among adult patients with community-acquired

Received 15 October 2015

pneumonia (CAP) has not been characterized yet. The aim of this study was twofold: rst,

Received in revised form

investigate the pathogen proles and the molecular epidemiology of respiratory viruses

27 December 2015

among Japanese CAP patients; and second, explore the clinical signicance of viral infections.

Accepted 4 January 2016

Methods: A cross-sectional observational study was conducted at Kyorin University Hospital.

To identify respiratory pathogens, hospitalized CAP patients were enrolled, and reverse

Keywords:

transcriptasepolymerase chain reaction technology was applied alongside conventional

Community-acquired pneumonia

microbiological methods. Phylogenetic and pairwise distance analyses of 10 viruses were

Pathogen prole

performed. CAP patients were divided into four etiological groups (virus alone, bacteria alone,

Respiratory virus

co-detection of virus and bacteria, and not detected) and the clinical ndings were compared.

Metapneumovirus

Results: Seventy-six patients were enrolled. Bacteria alone were detected in 39.5% (n30) of
CAP patients. Virus alone or co-detection were found in 10.5% (n8) and 11.8% (n 9) of cases,
respectively. Streptococcus pneumoniae and human metapneumovirus were the most frequently
detected bacterium and virus, respectively. Phylogenetic analyses of human metapneumovirus, human rhinovirus, and human respiratory syncytial virus showed that different
subgroups and genotypes might be associated with CAP. Respiratory failure was more
common when a virus was detected (both virus alone and co-detection groups; n17, 100%,
po0.05) than when a bacteria alone was detected (n17, 56.7%).
Conclusion: Prevalence of respiratory virus infection in CAP inpatients was 22.3%. The detected
viruses display high genetic divergence and correlate with increased respiratory failure.
& 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001

respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]

1.

Introduction

Community-acquired pneumonia (CAP) is a life-threatening

respiratory disease of worldwide importance [1]. According to
several studies from developed countries, the annual incidence
of CAP in adults is in the range of 0.51.1%, and the mortality
rate of hospitalized CAP patients is 414% [2]. Previous reports
have suggested that various pathogens including bacteria,
fungi, and viruses are associated with CAP [3]. Among these,
Streptococcus pneumoniae (pneumococcus) is a major cause of
CAP in adult patients, particularly in those with severe diseases
[25]. Bacteria such as Haemophilus inuenzae, methicillinsensitive Staphylococcus aureus (MSSA), Legionella pneumophila,
and Moraxella catarrhalis are associated with CAP in adult
inpatients [25], while other types such as Mycoplasma pneumoniae are detected in relatively mild CAP in outpatients [6].
Until recently, respiratory viruses such as human respiratory syncytial virus (RSV), human rhinovirus (HRV), human
parainuenza virus (HPIV), and human metapneumovirus
(HMPV) have been mainly associated with lower respiratory
tract infections (LRTI) including bronchitis, bronchiolitis, and
pneumonia in children [79].
Respiratory viruses such as RSV, HRV, HMPV, and adenovirus are present in two-thirds of children with CAP [10].
Apart from causing LRTI, these viruses can exacerbate asthma
and chronic obstructive pulmonary disease (COPD) [1114]. For
example, Dowell et al. showed that RSV was associated with 4.4%
of adult cases of LRTI during winter [15]. Recent molecular
epidemiological studies suggest that these viruses can be classied into numerous phylogenetic subtypes and genotypes. Specically, there are over 150 genotypes of HRV species A to C
(HRV-A to -C). Similarly, RSV and HPIV species can be subclassied into several genotypes. Recent studies have demonstrated
the presence of these respiratory viruses and/or bacteria in adult
patients with CAP [16,17]. However, the molecular epidemiology
in adult CAP Japanese patients is poorly understood.
We conducted pathogen proling and phylogenetic analyses of various respiratory viruses detected in hospitalized
CAP patients in Japan and characterized these patients in
terms of infectious viral and/or bacterial pathogens.

2.

Patients and methods

2.1.

Patients and study design

In this cross-sectional observational study, we recruited

consecutive patients admitted to Kyorin University Hospital

(Tokyo, Japan) between August 2012 and August 2014 with a

diagnosis of CAP. Pneumonia was dened as the presence of
new inltrates on chest X-rays along with other suggestive
signs and symptoms: cough, sputa, fever, chills, dyspnea,
pleuritic chest pain, disturbance of consciousness, and
crackles. Exclusion criteria included the following: (a) residence in a long-term nursing home or healthcare home;
(b) hospitalization within the preceding 90 days; (c) elderly
persons or physically disabled persons who needed healthcare; (d) continuous endovascular therapy (i.e., hemodialysis,
anti-cancer, or immunosuppressive drugs); (e) onset of pneumonia 48 h after admission; and (f) active tuberculosis.

2.2.

Clinical data collection

The following data were recorded on admission: age, sex,

comorbid illnesses (chronic heart diseases, COPD, asthma,
other lung diseases, diabetes mellitus, or active cancer),
immunodeciency status (i.e., use of immunosuppressive
drugs or prednisolone dose of Z5 mg/day, and HIV-positive
patients), use of anti-microbial drugs (anti-bacterial or antiinuenza) before admission, and clinical or laboratory ndings. Respiratory failure was dened as PaO2 o60 mmHg or
SpO2 o90% in room air. In patients who underwent home
oxygen therapy, respiratory failure was diagnosed at the
point when further oxygen supply was needed to maintain
the patients previous condition.
The severity of pneumonia was assessed using the pneumonia severity index (PSI), a prediction rule with points
assigned based on age, coexisting diseases, and abnormal
physical ndings. PSI straties CAP patients into ve classes
(IV) to predict their risk of mortality [18]. Severe pneumonia
is dened as PSI class IV or V. Follow-up variables included
the need for mechanical ventilation (invasive and non-invasive) within 5 days of admission, and mortality within
30 days.

2.3.

Samples

Samples collected on admission included sputum, nasopharyngeal swab (NPS), bronchoalveolar lavage uid (BALF), blood,
and urine. Serological tests for Mycoplasma pneumoniae were
performed on admission and after several weeks, when
possible. Invasive diagnostic methods were conducted according to clinical judgment. Respiratory samples for PCR-based
detection of respiratory viruses, Mycoplasma pneumoniae, and
Chlamydophila pneumoniae were collected separately from those

Corresponding author. Tel./fax: 81 422440671.

Corresponding author at: Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan. Tel.: 81 425610771; fax: 81 425908586.
E-mail addresses: kuraida@aol.com (D. Kurai), sasa-yosi@pref.gunma.lg.jp (Y. Sasaki), sara@yd5.so-net.ne.jp (T. Saraya),
h141@ks.kyorin-u.ac.jp (H. Ishii), tsuka-hiro@pref.gunma.lg.jp (H. Tsukagoshi), kkozawa-gi@umin.net (K. Kozawa),
aryo@yokohama-cu.ac.jp (A. Ryo), ishioka@nih.go.jp (T. Ishioka), makokuro@niid.go.jp (M. Kuroda), oishik@nih.go.jp (K. Oishi),
hajime@ks.kyorin-u.ac.jp (H. Takizawa), kimhiro@nih.go.jp (H. Kimura).
1
These authors contributed equally to this work.
nn

http://dx.doi.org/10.1016/j.resinv.2016.01.001
2212-5345/& 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001

respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]3

intended for bacterial culture and were stored at  80 1C

until use.

2.4.

Ethical approval

Samples were collected after written informed consent was

obtained from the subjects or their legal representatives. The
study protocol was approved by the Ethics Committee on
Human Research of Kyorin University Hospital (H24-021) on
July 31, 2012. The protocols were carried out in accordance
with approved guidelines.

2.5.

Bacteriological examination

Acceptable sputum and BALF samples were cultured on 5%

blood agar, chocolate agar, and modied Conradi-Drigalski
agar for the isolation and identication of bacterial pathogens. Blood cultures were incubated under aerobic and
anaerobic conditions using the BacT/Alert system (bioMrieux, Marcy, l'Etoile, France). Urine samples were used for the
detection of pneumococcus and L. pneumophila antigens using
BinaxNOWs (Alere Medical Co., Ltd., Shinjuku, Japan).

2.6.
RNA extraction, PCR, and gene sequencing of the
pathogens
Samples were centrifuged at 3000g at 4 1C for 30 min. Viral
RNA and DNA were extracted from supernatants using the
QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA).
Reverse transcription was performed using PrimeScript RT
reagent Kit (Takara Bio, Otsu, Japan), according to the manufacturers instructions. Using PCR, we aimed to detect various
respiratory viruses such as HMPV, HRV, enterovirus, RSV,
inuenza viruses A, B, and C (InfV-A, B, and C), HPIV, human
coronavirus, adenovirus, Mycoplasma pneumoniae, Chlamydophila pneumoniae, cytomegalovirus (CMV), human parvovirus
B19, varicella zoster virus, and human bocavirus as described
previously [1926]. PCR products were puried using MonoFas
DNA Purication Kit I (GL Sciences Inc., Shinjuku, Tokyo,
Japan). The puried products were sequenced with a BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,
Foster City, CA, USA) using the above primers [1926].
Sequence analysis was performed on an ABI 3130 Genetic
Analyzer (Applied Biosystems). The nucleotide sequences thus
obtained were given GenBank accession numbers from
LC020476 to LC020488.

2.7.

Diagnostic criteria of causative pathogens

We considered that infection was caused by a specic

pathogen if one or more of the following criteria were met.
(1) Positive bacterial culture was obtained from acceptable
sputum samples with a predominant species and compatible
results from Gram staining. Samples were considered acceptable when 425 polymorphonuclear cells were observed
under low-power magnication [27]. (2) The pathogen was
cultured from blood samples and, except for the lungs, no
other organs were known to be involved. (3) A urinary antigen
test was positive for pneumococcus or L. pneumophila. (4) A
fourfold increase in complement xation titers for

Mycoplasma pneumoniae was detected from paired sera.

Finally, (5) respiratory viruses Mycoplasma pneumoniae or
Chlamydophila pneumoniae were detected in respiratory samples by PCR [28]. CAP patients were classied into 4 etiological
groups: virus alone, bacteria alone, co-detection of virus and
bacteria, and not detected. Clinical characteristics including
respiratory failure were compared between the groups.

2.8.
Phylogenetic analyses by the neighbor-joining (NJ)
method and genotyping of HMPV, RSV, and HRV
We performed phylogenetic analyses using Molecular Evolutionary Genetics Analysis (MEGA) software, version 5.0. Phylogenetic analysis of HMPV, RSV, and HRV were based on
parts of the F gene (317 bp), G gene (240312 bp on RSV-A, 234
294 bp on RSV-B), and VP4/VP2 coding region (390 bp), respectively. Evolutionary distances were estimated using Kimura
two-parameter method, and phylogenetic trees were constructed using the NJ method. Reliability of the trees was
estimated using 1000 bootstrap replications.

2.9.
Calculation of pairwise distances of detected
respiratory viruses
We calculated pairwise distances (p-distance) of the HMPV,
RSV, and HRV species detected in this study using MEGA
software, version 5.0. Calculations were based on the nucleotide sequence of each virus, as described in Section 2.8.

2.10.

Statistical analysis

Data was analyzed using StatView 5.0 (SAS Institute, Cary,

NC, USA). Differences in proportions between the groups
were assessed using Fishers exact test (2 groups) and the
chi-squared test (4 groups). Other comparisons between
groups were done using MannWhitney U (2 groups) and
KruskalWallis (4 groups) tests. Statistical signicance was
dened by a two-sided -level of 0.05.

3.

Results

3.1.

Data obtained from patients

Seventy-six patients were enrolled in this study. Forty-eight

respiratory samples (sputum n 46, BALF n 2) were acceptable for bacterial culture and identication. Respiratory
samples (NPS n 59, BALF n 2, sputum n 15) were used to
detect viral and atypical pathogens by PCR-based methods.
Urine antigen tests and blood cultures were examined in 66
and 72 patients, respectively.

3.2.

Pathogen proles of inpatients with CAP

In this study, 76 CAP inpatients were enrolled between August

2012 and August 2014, as detailed in Tables 1 and 2. Bacteria
alone were detected in 30 patients (39.5%). Pneumococcus was
the most frequently detected bacterium (n 11, 14.5%) followed
by Mycoplasma pneumoniae (n 4), MSSA (n 4), H. inuenzae (n 2),
M. catarrhalis (n2), and Streptococcus anginosus (n 2). Neither

Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001

respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]

Table 1 Summary of patient characteristics.

Number of patients
Agen
Male

Severe pneumonia (PSI class IV or V)

Wheezing
Respiratory failure
Mechanical ventilation
(Invasive mechanical ventilation)
Mortality within 30 days#

Duration of respiratory failure (days)

Laboratory ndings
White blood cell count, 106 cells/mLn
C-reactive protein, mg/dLn
Procalcitonin, ng/mLn

All patients

Virus alone

Bacteria alone

Co-detection

Not detected

p value

76 (100)
71.5 (58.378.0)
51 (67.1)

8 (10.5)
76.5 (69.578.0)
4 (50.0)

30 (39.5)
67.5 (48.874.8)
22 (73.3)

9 (11.8)
68.0 (64.077.0)
5 (55.6)

29 (38.2)
73.0 (52.078.0)
20 (69.0)

NS
NS

39 (51.3)

20
51
7
5
3/64

(26.3)
(67.1)
(9.2)
(6.6)
(4.7)

5.0 (010.0)

11.1 (7.915.4)
11.4 (4.123.1)
0.39 (0.092.46)

Comorbidity
Asthma
COPD
Chronic heart disease
Immunodeciency
Diabetes mellitus
Malignancy
Other lung disease

8
20
18
10
13
4
8

Antimicrobials
Anti-bacterial drug
Anti-inuenza drug

22 (28.9)
4 (5.3)

(10.5)
(26.3)
(23.7)
(13.2)
(17.1)
(5.3)
(10.5)

4 (50.0)

2
8
1
0
0/8

15 (50.0)

(25.0)
(100) a,
(12.5)
(0)
(0)

7.0 (5.011.3)

7.7 (6.89.7)
4.1 (2.811.3)
0.13 (0.060.60)

1
2
3
0
2
0
1

(12.5)
(25.0)
(37.5)
(0)
(25.0)
(0)
(12.5)

5 (62.5)
1 (12.5)

8
17
2
1
1/22

a, c

3.0 (08.8)

(10.0)
(30.0)
(16.7)
(6.7)
(20.0)
(10.0)
(10.0)

4 (13.3)e,
1 (3.3)

5
9
2
2
1/8

(55.6)
(100) c,
(22.2)
(22.2)
(12.5)

14 (48.3)

7.0 (4.016.0)

13.1 (9.015.3)
13.0 (6.822.0)
0.80 (0.112.84)

3
9
5
2
6
3
3

e, f

(26.7)
(56.7)
(6.7)
(3.3)
(4.5)

6 (66.7)

9.4 (5.815.3)
6.6 (2.720.2)
0.29 (0.094.89)

3
2
1
2
1
0
0

(33.3)
(22.2)
(11.1)
(22.2)
(11.1)
(0)
(0)

0 (0)f,
0 (0)

5
17
2
2
1/26

(17.2)
(58.6)
(6.9)
(6.9)
(3.8)

NS

b, d

3.0 (010.0)

11.1 (8.615.6)
17.1 (2.725.9)
0.41 (0.110.94)

1
7
9
6
4
1
4

(3.4)
(24.1)
(31.0)
(20.7)
(13.8)
(3.4)
(13.8)

13 (44.8)g,
1 (3.4)

NS
o0.05
NS
NS
NS

NS

NS
NS
NS

NS
NS
NS
NS
NS
NS
NS

o0.05
NS

Data are expressed as number (percentage) unless otherwise stated.

Data are expressed as median (IQR).
#
Data are expressed as number of deaths/n (percentage) and missing date was excluded. NS: not signicant.
a
p 0.035: virus alone vs bacteria alone.
b
p 0.036: virus alone vs not detected.
c
p 0.018: co-detection vs bacteria alone.
d
p0.036: co-detection vs not detected.
e
p 0.010: virus alone vs bacteria alone.
f
p0.010: virus alone vs co-detection.
g
p 0.016: bacteria alone vs not detected.
h
p 0.010: co-detection vs not detected.
n

Mycoplasma pneumoniae nor Chlamydophila pneumoniae were

detected by PCR. All patients infected with Mycoplasma pneumoniae were diagnosed using paired sera.
In 8 of the 76 patients (10.5%), viral infections alone were
detected; they included HMPV (n 3), CMV (n 2), RSV (n 1),

respiratory failure on days 3 and 17, while the third died on

day 2 from severe sepsis.

3.3.
Proportion of patients with severe pneumonia among
the groups

InfV-A (n 1), and HPIV (n 1). Furthermore, bacterial and

viral co-detection was found in 9 (11.8%) patients, and in all
those with HRV, where the main pathogenic pattern was
HRVpneumococcus (n 2). No pathogens were detected in
29 (38.2%) patients. Three patients (MSSA/Pseudomonas aeruginosa, n 1; unknown, n 1; HRV/pneumococcus, n 1) died
within 30 days of admission. The rst two patients died of

No signicant differences were found between the etiological

groups (virus alone, bacteria alone, co-detection, or not
detected) and the proportions of patients with severe pneumonia (PSI IV or V) in each of them (Table 1).
However, signicant differences between the groups were
found in relation to rates of respiratory failure and antibiotics use.

Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001

respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]5

Table 2 Distribution of detected pathogens.

Virus alone
HMPV
CMV
RSV
InfV-A
HPIV
Bacteria alone
Pneumococcus
M. pneumoniae
MSSA
M. catarrhalis
H. inuenzae
S. anginosus
P. aeruginosa
E. coli
S. marcescens
PneumococcusH. inuenzae
P. aeruginosaMSSA
Virusbacteria
HRV
Pneumococcus
Group G Streptococcus
PneumococcusH. inuenzae
HMPV
Pneumococcus
MSSA
PneumococcusH. inuenzae MSSA
RSV
Pneumococcus
CMVpneumococcus

8
3
2
1
1
1
30
11
4
4
2
2
2
1
1
1
1
1
9
2
1
1
1
1
1
1
1

HMPV: human metapneumovirus; CMV: cytomegalovirus; RSV:

respiratory syncytial virus; InfV-A: inuenza virus A; HPIV: human
parainuenza virus; HRV: human rhinovirus; Pneumococcus:
Streptococcus pneumoniae; MSSA: methicillin sensitive Staphylococcus
aureus.

3.4.
Virus-positive (virus alone and co-detection groups)
vs bacteria alone groups
Among CAP patients, the frequency of respiratory failure was
more common when a virus was detected (both virus only
and co-detection groups, n 17, 100%, po0.05) than in bacteria alone cases (n 17, 56.7%; Table 1).
Respiratory failure in the virus-positive groups (virus
alone, median 7, interquartile range (IQR) 511.3 days; codetection, median 7, IQR 416 days) lasted longer than in the
bacteria alone group (median 3, IQR 08.8 days).

3.5.

Co-detection group vs bacteria alone group

The co-detection group (100%) was more susceptible to

respiratory failure than the bacteria alone group (56.7%,
po0.05). The combinations of pathogens found in the codetection group are shown in Table 2. The need for invasive
mechanical ventilation was higher in the co-detection group
(n 2, 22.2%) than in the bacteria alone (Mycoplasma pneumoniae) group (n 1, 6.7%), but this result was not statistically
signicant (p 0.06). The combinations of causative

pathogens in the co-detection group were HRVpneumococcus (n 1), and HRVpneumococcusH. inuenzae (n 1).

3.6.
Genetic and phylogenetic properties of respiratory
viruses detected in CAP inpatients
We constructed phylogenetic trees based on the nucleotide
sequences of genes from the various viruses. Phylogenetic
trees constructed by the NJ method are shown in Fig. 1ac.
The most commonly detected virus was HMPV (HMPV alone,
3 patients; HMPVbacteria, 3 patients). Phylogenetic analysis
conrmed the HMPV subgroups to be A2 (2 strains), B1
(1 strain), and B2 (3 strains), which may be prevalent in Japan.
Three RSV strains (RSV alone, 1 patient; RSVbacteria, 2
patients) were detected among inpatients, and their genotypes were conrmed as ON1 (1 strain) and BA9 (2 strains). In
addition, 4 HRV strains (HRVbacteria) were detected, all of
which belonged to HRV-A genotypes (HRV-A1, HRV-A29,
HRV-A103, and HRV-A71).
The p-distance values between the detected HMPV, RSV,
and HRV strains were 0.09670.061, 0.4770.40, and 0.2270.031,
respectively. These results indicate that the viruses belonged
to various subgroups and genotypes, and the strains displayed
relatively high genetic diversity.

4.

Discussion

We conducted pathogen proling of Japanese inpatients (76

cases) with CAP and performed genetic analyses of the
various respiratory viruses detected such as HMPV, RSV,
and HRV. Bacteria alone were detected in 39.5% patients (30
cases), while viruses alone were detected in 10.5% patients (8
cases). Co-detection was noted in 11.8% patients (9 cases).
Pneumococcus and HMPV were the most commonly detected
bacterium and virus, respectively. In addition, phylogenetic
analyses of HMPV, RSV, and HRV indicate that different
subgroups and genotypes of the viruses may be associated
with CAP inpatients in Japan.
Many previous reports have suggested that pneumococcus
is a major causative pathogen in CAP [25,28]. For example,
Lim et al. showed that pneumococcus was the predominant
causative bacterium in hospitalized CAP patients in the
United Kingdom, followed by Chlamydophila pneumoniae and
H. inuenzae [29]. Ishida et al. reported that the bacterial
pathogens in Japanese CAP patients were pneumococcus,
H. inuenzae, and Mycoplasma pneumoniae, in descending order
of prevalence [30]. The detection frequencies of the isolated
bacteria were similar to those in the present study.
Few studies have been reported on pathogen proles,
including respiratory viruses, in adult Japanese CAP patients
[31]. In addition, the molecular epidemiology of CAPassociated viruses is poorly understood; although, some
CAP-associated respiratory viruses such as HMPV and HRVC have recently been conrmed [3234]. Our results show that
HMPV, RSV, HRV, and HPIV are associated with CAP in adult
inpatients. The viruses were classied into subgroups and
genotypes based on genetic divergence in the phylogenetic
trees (Fig. 1ac). This indicates that viruses such as HMPV,
RSV, and HPIV could have been associated with over 10% of

Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001

respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]

Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001

respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]7

CAP inpatients. Although we found no correlation between

the phylogenies of these viruses and disease severity, the
estimate was inferred from the seasonal prevalence of these
strains [35]. Regarding CMV detection, it is not possible to
distinguish between active respiratory infection and asymptomatic/latent infection, especially in clinically ill patients
[36,37].
The present study conrmed two previous observations.
First, bacteria (alone) were the most frequently detected
pathogens among CAP patients (39.5%), and there was equal
prevalence of virus only (10.5%) and co-detection (11.8%), as
reported before [28,38,39]. Thus, viral infections should be
considered major pathogens of CAP, even in adult patients.
Second, respiratory failure on admission was signicantly
more common (po0.05) and lasted longer in the virus or codetection groups than in the bacteria alone group. Similarly,
Johansson et al. reported that co-infected CAP patients
suffered from tachypnea and required oxygen therapy over
a longer period compared with subjects infected with bacteria
alone [40].
Bacterial and viral co-infections may increase the severity
of pneumonia. Several mechanisms have been proposed such
as alteration of the host immune response, deterioration of the
respiratory status following co-infection with pneumococcus
and inuenza virus in mice [41], and increased susceptibility to
bacterial (pneumococcus) and/or viral infection (HMPV) caused
by previous viral (HMPV/inuenza virus) infections in mice
and in vitro [42,43].
Here, we show that 2 of the 4 patients in the co-detection
group with HRV and bacteria (pneumococcus or pneumococcusH. inuenzae) required mechanical ventilation. Previous studies reported that HRV caused secondary bacterial
infections in up to 60% of COPD patients due to cleavage of
antimicrobial peptides, secretory leukoprotease inhibitor or
elan, by virus-induced neutrophil elastase, supporting a
hypothesis that co-detection (HRVbacteria) itself is vulnerable to more severe respiratory status. [4446]. Here, bacteria
were co-detected in all 4 HRV patients, which might explain
the high frequency of invasive mechanical ventilation.
Comorbidity may have an impact on the etiology of CAP;
some studies have shown that patients with cardiovascular

disease are vulnerable to viral CAP [47,48]. However, we found

that the proportions of comorbid illnesses (i.e., COPD,
asthma, and chronic heart disease) did not differ between
groups (virus alone, bacteria alone, co-detection) (Table 1).
Thus, we conclude that viral and bacterial co-infection
could lead to deterioration of respiratory status in adult CAP
patients.
The rate of detection of inuenza virus in CAP patients
(1.3%) was lower than previously reported (4.49.5%) [14,28,39].
This might reect the high frequency of neuraminidase
inhibitors (NAIs) administered to patients with inuenza in
Japan [49].
Our study had the following limitations. First, it was
conducted at a single tertiary center in the west side of
Tokyo; hence, results may be affected by both anti-bacterial
and anti-inuenza drugs prescribed prior to admission by
doctors in that area. Second, seasonal inuenza virus was
detected in only one case. This may be a result of the
characteristic facilities of a university hospital. Hence, there
may have been some bias in the selection of the present
subjects.
Finally, upper respiratory samples, such as NPS, were used
for viral detection in this study. It has been suggested that
some respiratory viruses such as HRV and HMPV can be
detected in these samples in up to 2.1% of healthy adults
[17,50]; however, we did not examine healthy adult controls.
Self et al. reported that any respiratory virus could be
associated with adult CAP; therefore, the viruses detected in
this study were possible pathogens.
To the best of our knowledge, this is the rst genetic and
phylogenetic investigation of viral infections in Japanese
adult CAP patients.

5.

Conclusion

This study demonstrates that respiratory viruses (i.e., HMPV,

RSV, or HRV) displaying high genetic divergence are present
in approximately 22.3% of adult CAP patients in whom viral
infections can accelerate respiratory failure.

Fig. 1 (a) Phylogenetic tree of HMPV nucleotide sequences based on F gene (317 bp). The phylogenetic tree was constructed
by the neighbor-joining method using MEGA software 5.0. Distances were calculated according to Kimuras two-parameter
method. Bootstrap values Z80% are shown at the branch nodes. The tree includes the present strains (n 6) and reference
strains (n 12). The following references strains were used: NL/00/1 (AF371337), CAN99-81 (M18759), NL/17/00 (AY304360),
CAN97-83 (AY145296), O0601 (EF589610), JPS03-240 (AY530095), NL/1/99 (AY304361), JPS03-194 (AY530094), CAN97-82
(EU814623), JTY06-1 (EU127917), CAN98-75 (M18761), and NL/1/94 (AY304362). (b) Phylogenetic tree of RSV nucleotide
sequences based on the G gene (240312 bp). The phylogenetic tree was constructed by the neighbor-joining method using
MEGA software 5.0. Distances were calculated according to Kimuras two-parameter method. Bootstrap values Z80% are
shown at the branch nodes. The tree includes the present strains (n 3) and reference strains (n 20). The following references
strains were used: A2 (M11486), Long (AY911262), AL19452-2 (AF233901), NY20 (AF233918), MO02 (AF233910), SA99V1239
(AF348808), LLC242-282 (AY114150), NY_CH09_93 (AF065254), MO55 (AF233915), LLC235-267 (AY114149), NG-016-04
(AB470478), MY-2444006-11 (JX256871), 18537 (M17213), Ken_2_00 (AY524575), NY01 (AF233931), CH93_9b (AF065251), MO35
(AF233929), BA4128_99B (AY333364), RSV/YOK/07/4 (AB551076), and NG-040-07 (AB470478). (c) Phylogenetic tree for HRV-A
nucleotide sequences based on VP4/VP2 coding region (390 bp). The phylogenetic tree was constructed by the neighborjoining method using MEGA software 5.0. Distances were calculated according to Kimuras two-parameter method. Bootstrap
values Z80% are shown at the branch nodes. The tree includes the present strains (n 4) and reference strains (n 12). The
sequences of the reference strains were obtained from picornaviridae.com (URL: http://www.picornaviridae.com/).
Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001

respiratory investigation ] (] ] ] ]) ] ] ] ] ] ]

Conict of interest
The authors have no conicts of interest.

Authors contributions
DK, TS, HI, HTs, AR, KO, HT, and HK designed the research;
DK, YS, TS, HTs, KK, and TI performed the research; YS, HTs,
MK, and KO contributed analytic tools; DK, YS, TS, and HK
analyzed data; DK, YS, TS, HT, and HK wrote the paper.

Acknowledgments
This work was supported by a Grant-in-Aid from the Japan
Society for the Promotion of Science and for Research on
Emerging and Re-emerging Infectious Diseases (H25-ShinkoShitei-001) from the Japanese Ministry of Health, Labour and
Welfare.

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Please cite this article as: Kurai D, et al. Pathogen proles and molecular epidemiology of respiratory viruses in Japanese
inpatients with community-acquired pneumonia. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.
resinv.2016.01.001